22 research outputs found

    Phycobilisome rod mutants in Synechocystis sp. strain PCC6803

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    The phycobilisome is a large pigment-protein assembly that harvests light energy for photosynthesis. This supramolecular complex is composed of two main structures: a core substructure and peripheral rods. Linker polypeptides assemble phycobiliproteins within these structures and optimize light absorption and energy transfer. Mutations have been constructed in three rod-linker-coding genes located in the cpc operon of Synechocystis sp. strain PCC6803. The cpcC1 gene encoding the 33 kDa linker is found to be epistatic to cpcC2 encoding the 30 kDa linker, indicating a specific role for each of these two linkers in rod growth. This corroborates studies on the sequential degradation of phycobilisomes upon nitrogen starvation. Three allelic mutants affecting cpcC2 revealed a polar effect of commonly used cassettes (aphI, aadA) on the operon steady-state transcripts and an effect of rod linker availability on the amount of phycocyanin incorporated in the phycobilisome. This led to the proposal that regulation of rod length could occur through processing of transcripts upstream of the cpcC2 gene

    Phycobilisome linker proteins are phosphorylated in Synechocystis sp. PCC 6803

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    The controversial issue of protein phosphorylation from the photosynthetic apparatus of Synechocystis sp. PCC 6803 has been reinvestigated using new detection tools that include various immunological and in vivo labeling approaches. The set of phosphoproteins detected with these methods includes ferredoxin-NADPH reductase and the linker proteins of the phycobilisome antenna. Using mutants that lack a specific set of linker proteins and are affected in phycobilisome assembly, we show that the phosphoproteins from the phycobilisomes correspond to the membrane, rod, and rod-core linkers. These proteins are in a phosphorylated state within the assembled phycobilisomes. Their dephosphorylation requires partial disassembly of the phycobilisomes and further contributes to their complete disassembly in vitro. In vivo we observed linker dephosphorylation upon long-term exposure to higher light intensities and under nitrogen limitation, two conditions that lead to remodeling and turnover of phycobilisomes. We conclude that this phosphorylation process is instrumental in the regulation of assembly/disassembly of phycobilisomes and should participate in signaling for their proteolytic cleavage and degradation

    Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light

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    Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δflv1(A) and/or Δflv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis

    A gene regulation mechanism that allows synthesis of two ferredoxin (NADP oxidoreductase isiforms from a single gene in the cyanobacterium Synechocystis sp. PCC 6803)

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    La Ferrédoxine:NADP oxydoréductase (FNR), codée par le gène petH, est une enzyme qui catalyse la production du NADPH dans les cellules photoautotrophes, ainsi que sa consommation dans les cellules hétérotrophes. Alors que le gène petH est unique chez la cyanobactérie Synechocystis sp. PCC6803, deux isoformes de FNR sont synthétisées FNRL et FNRS. Il a été montré que FNRS était le produit d'une initiation de traduction interne dans le même cadre de lecture que FNRL. Durant ma thèse, j'ai découvert un mécanisme grâce auquel un gène peut coder deux isoformes. Tout d'abord, j'ai montré que chaque isoforme pouvait être codée par un transcrit spécifique. En effet, en conditions standards (où FNRL s'accumule) deux ARNm avec des séquences "leader" similaires (32 et 53 bases) sont transcrits alors qu'en conditions de carence en azote (où FNRS s'accumule) un ARNm portant une séquence "leader" plus longue (126 bases) est transcrit. Des fusions transcriptionnelles du promoteur lac de Escherichia coli avec les différentes régions transcrites de petH, nous ont montré que cette régulation pourrait être accomplie par des structures secondaires adoptées par la région leader des ARNm. De telles structures pouvant activer l'initiation de traduction de FNRS et inhiber celle de FNRL. La cartographie des complexes d'initiation de traduction montre que dans l'ARNm le plus long le complexe se trouve dans la région initiatrice de FNRL, alors que dans les ARNm plus courts celui-ci est localisé dans la région initiatrice de FNRS. Ainsi, nous avons mis en évidence un nouveau mécanisme de régulation génétique chez Synechocystis permettant la synthèse de deux isoformes à partir d'un seul gèneFerredoxin:NADP oxidoreductase (FNR), encoded by the petH gene, provides NADPH for CO2 fixation in photoautotrophic cells and oxidizes NADPH in heterotrophic cells. Whereas there is only one petH gene copy in the cyanobacterium Synechocystis sp. PCC6803, two FNR isoforms accumulate (FNRL and FNRS). It was proposed that FNRL fulfills functions in linear electron transport while FNRS is involved in cyclic electron transport and respiration. In addition, FNRS was shown to be the product of an internal translation initiation within the FNRL open-reading frame. During my PhD, I revealed the mechanism by which petH translation leads to the accumulation of either one of the FNR isoforms. I showed that each isoform is produced from a specific mRNA; under standard conditions -when FNRL accumulates- two mRNAs carrying similar leaders (32 and 53 bases) are transcribed; while under nitrogen starvation -when FNRS accumulates- an mRNA, carrying a longer leader (126 bases), is transcribed. Transcriptional fusions of the Escherichia coli lac promoter to petH, with different leader sequences, suggest that the translation regulation does not require a specific factor; but rather to a spontaneously occurring secondary structure, adopted by the longer leader. Such a structure could activate FNRS translation initiation and prevent that of FNRL. Toeprinting assays confirmed this hypothesis in vitro; translation initiation complexes locations were mapped to the FNRL initiator codon in the short mRNA and to the FNRS initiator codon in the longer mRNA. Thus we have uncovered a novel gene-regulation mechanism by which two isoforms are produced from a single gene in Synechocystis sp. PCC 6803PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

    Cyclic Electron Flow-Coupled Proton Pumping in Synechocystis sp. PCC6803 Is Dependent upon NADPH Oxidation by the Soluble Isoform of Ferredoxin:NADP-Oxidoreductase

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    International audienceFerredoxin:NADP-oxidoreductase (FNR) catalyzes the reversible exchange of electrons between ferredoxin (Fd) and NADP(H). Reduction of NADP+ by Fd via FNR is essential in the terminal steps of photosynthetic electron transfer, as light-activated electron flow produces NADPH for CO2 assimilation. FNR also catalyzes the reverse reaction in photosynthetic organisms, transferring electrons from NADPH to Fd, which is important in cyanobacteria for respiration and cyclic electron flow (CEF). The cyanobacterium Synechocystis sp. PCC6803 possesses two isoforms of FNR, a large form attached to the phycobilisome (FNRL) and a small form that is soluble (FNRS). While both isoforms are capable of NADPH oxidation or NADP+ reduction, FNRL is most abundant during typical growth conditions, whereas FNRS accumulates under stressful conditions that require enhanced CEF. Because CEF-driven proton pumping in the light–dark transition is due to NDH-1 complex activity and they are powered by reduced Fd, CEF-driven proton pumping and the redox state of the PQ and NADP(H) pools were investigated in mutants possessing either FNRL or FNRS. We found that the FNRS isoform facilitates proton pumping in the dark–light transition, contributing more to CEF than FNRL. FNRL is capable of providing reducing power for CEF-driven proton pumping, but only after an adaptation period to illumination. The results support that FNRS is indeed associated with increased cyclic electron flow and proton pumping, which is consistent with the idea that stress conditions create a higher demand for ATP relative to NADPH

    Overexpression of plastid terminal oxidase in Synechocystis sp. PCC 6803 alters cellular redox state

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    Cyanobacteria are the most ancient organisms performing oxygenic photosynthesis, and they are the ancestors of plant plastids. All plastids contain the plastid terminal oxidase (PTOX), while only certain cyanobacteria contain PTOX. Many putative functions have been discussed for PTOX in higher plants including a photoprotective role during abiotic stresses like high light, salinity and extreme temperatures. Since PTOX oxidizes PQH2 and reduces oxygen to water, it is thought to protect against photo-oxidative damage by removing excess electrons from the plastoquinone (PQ) pool. To investigate the role of PTOX we overexpressed rice PTOX fused to the maltose-binding protein (MBP-OsPTOX) in Synechocystis sp. PCC 6803, a model cyanobacterium that does not encode PTOX. The fusion was highly expressed and OsPTOX was active, as shown by chlorophyll fluorescence and P700 absorption measurements. The presence of PTOX led to a highly oxidized state of the NAD(P)H/NAD(P)(+) pool, as detected by NAD(P)H fluorescence. Moreover, in the PTOX overexpressor the electron transport capacity of PSI relative to PSII was higher, indicating an alteration of the photosystem I (PSI) to photosystem II (PSII) stoichiometry. We suggest that PTOX controls the expression of responsive genes of the photosynthetic apparatus in a different way from the PQ/PQH2 ratio.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'
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